Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is the causative agent for Kaposi sarcoma (KS)1,2 and is also associated with primary effusion lymphoma (PEL)3 and multicentric Castleman disease (MCD).4 In northern Europe and the United States, KSHV infection is rare apart from that occurring in men who have sex with men (MSM), among whom transmission is thought to be largely sexual.5 In sub-Saharan Africa, where KS is endemic, KSHV is highly prevalent and infection occurs in children and adults6 in a pattern similar to that found in the Mediterranean region.7,8 In most studies, KSHV prevalence in children increases with age, suggesting that nonsexual horizontal transmission plays an important role.6-8 Children born to KSHV-seropositive mothers are at higher risk of being KSHV-seropositive,9,10 and KSHV infection rates in children have been correlated with higher latent KSHV antibody titers in mothers.11 The evidence for vertical transmission during birth or by breast milk is weak, however.12,13 KSHV transmission to children within families has also been reported,14,15 further supporting horizontal transmission of the virus.
Few studies have investigated the effects of other factors on KSHV infection in children. Poor hygiene practices, lower socioeconomic status, and environmental factors such as the source of drinking water may play a role.16 KSHV- and HIV-coinfected subjects are at an especially high risk of developing KS.17 There have been reports of increases in KS incidence among HIV-negative individuals in Africa,18 leading to speculation that KSHV may be spread more readily from HIV-positive individuals to the HIV-negative population. The effects of coinfection of mothers with HIV and KSHV on KSHV transmission to children are not well understood. In this study, we assessed the connection between KSHV seropositivity in children in relation to their mother's KSHV status using 2 serologic assays. In addition, we assessed whether KSHV and HIV coinfection in mothers was associated with higher KSHV seropositivity in their children.
MATERIAL AND METHODS
The National Health Laboratory Service (NHLS), Department of Human Genetics, in Johannesburg collects blood from branch laboratories across all 9 provinces of South Africa from mothers, their children, and putative fathers to resolve cases of disputed paternity. During September 1999 through May 2001, the NHLS Cancer Epidemiology Research Group consecutively collected leftover blood samples from 1179 mothers and their 1287 confirmed children. Deidentified information was obtained about the age of mothers and age and gender of children. The study was approved by the University of the Witwatersrand Research Ethics Committee (Medical). The mean ages (SD) of all mothers and children were 30.0 (7.1) and 5.5 (4.9) years, respectively. Age was not significantly different between male and female children (P = 0.56).
Serum samples were stored at −20°C and shipped on dry ice to the Viral Epidemiology Section, AIDS Vaccine Program, Science Applications International Corporation (SAIC), National Cancer Institute (NCI), Frederick, Maryland, for KSHV antibody testing. KSHV infection was determined using 2 enzyme-linked immunoassays (ELISAs). One detected antibodies to the KSHV K8.1 glycoprotein, and the other detected KSHV antibodies to latent KSHV ORF73. These methods are described in general elsewhere.19,20
Additional quality control measures were included for this study because of the large sample size. Three positive and 3 negative standard controls were included on each plate, and a titration of positive controls was also included for each batch of 6 plates. Diluted sera from South African subjects with KS, previously confirmed to be KSHV-seropositive by a latent KSHV ORF73 immunofluorescence assay,17 were tested as additional controls within the analyzed batches.
The cutoff points for each assay were previously empirically defined using a panel of well-characterized samples from patients with AIDS and patients with classic KS, MSM, HIV-infected subjects with hemophilia, and blood donors (data not shown). Cutoff points were adjusted to take account of plate-to-plate and day-to-day variation by including variation observed in the positive or negative controls in the calculation. The cutoff point for the LANA ORF73 assay was calculated as the mean of the positive controls (MPC) per plate divided by 5, because most variation was seen with the positive controls in this assay. For the lytic K8.1 assay, most variation was seen in the negative controls; therefore, the cutoff was defined as the mean of the negative controls (MNC) per plate plus 0.75.
HIV antibody status was determined at the NHLS Contract Research Laboratories, Johannesburg, South Africa, using the Abbott IMX System HIV-1/HIV-2 III enzyme immunoassay kit (Abbott Laboratories, Abbott Park, IL). The laboratory technicians who performed all the laboratory analyses were unaware of subjects' characteristics.
We analyzed data using the SAS 9.1 statistical package (SAS Institute, Cary, NC). Correlations between the 2 KSHV assays were estimated using the Pearson correlation coefficient. We assessed associations between KSHV seropositivity of mothers and children using the χ2 test for homogeneity and for trend. Analyses of association of KSHV with other factors were restricted to those subjects aged 1.6 and 10 years. We estimated odds ratios (ORs) and 95% confidence intervals (CIs) using logistic regression models adjusted for the age of the mother (≤25, 26-29, 30-34, and ≥35 years) and the age of the child (1.6-3, 4-6, and 7-10 years). Children aged 18 months or younger were analyzed separately because of the possible presence of maternal antibodies, and children older than 10 years of age were also analyzed separately because of possible sexual transmission. Using optical density (OD), we grouped all mothers who were KSHV-seronegative into a “low” antibody titer category and divided KSHV-seropositive mothers into 2 groups, “medium” and “high” antibody titers, based on measurements being greater than or less than the median of the positives. Because the distribution of the lytic K8.1 and latent ORF73 values was skewed, we log-transformed the OD readings and reported geometric means and SD ranges. All P values were 2-sided. We considered P < 0.05 to be statistically significant. We recognize that there might be some degree of clustering of infections among mothers who had multiple children; however, the influence of this did not make any material difference to the results.
Determination of Kaposi Sarcoma-Associated Herpesvirus Seropositivity
There was moderate concordance between the lytic KSHV K8.1 and latent KSHV ORF73 assays in detecting KSHV seropositivity (κ = 0.44) and a significant correlation between the ODs for the 2 assays (r = 0.45, P < 0.0001). KSHV seroprevalence refers to being seropositive to antibodies to lytic KSHV K8.1 or latent KSHV ORF73/LANA antigens.
KSHV Seroprevalence and Associations Between Kaposi Sarcoma-Associated Herpesvirus Seropositivity in Mothers and Their Children
A total of 554 mothers and children were seropositive for KSHV on the lytic KSHV K8.1 or latent KSHV ORF73 assay, and 200 of 554 were positive on both assays. The seroprevalence of KSHV among the 1287 children was 15.9% (Table 1). Prevalence did not differ by gender (P = 0.12) and did not increase with age (Ptrend = 0.43). The seroprevalence of KSHV in the 1179 mothers was 29.7% (see Table 1B) and, in contrast, did increase with age (Ptrend = 0.03).
Table 2 shows the relation between KSHV status in mothers and their children. Overall, among children aged 1.5 to 10 years, children of KSHV-seropositive mothers had a 2-fold increased risk of KSHV seropositivity compared with children of KSHV-seronegative mothers (OR = 1.9, 95% CI: 1.3 to 3.0). The odds of infection in children peaked at almost 3-fold in children aged 7 to 10 years (OR = 2.8, 95% CI: 1.4 to 5.7). Among children 18 months of age or younger, the seroprevalence of KSHV was 16.4% in children of KSHV-negative mothers and 21.5% in children of KSHV-seropositive mothers. In this young age group, however, the mothers' KSHV status was not associated with an increased risk of KSHV seropositivity in their children (OR = 1.5, 95% CI: 0.8 to 2.7). In children aged 11 to 16 years, 14.6% of children of KSHV-seronegative mothers were KSHV-seropositive, whereas 21.8% of children of KSHV-seropositive mothers were KSHV-seropositive (OR = 1.7, 95% CI: 0.7 to 4.1). Seropositivity to latent ORF73 antibodies in children was not associated with the mothers' ORF73 antibody level (Ptrend = 0.12). The risk of lytic K8.1 seropositivity in children increased with increased levels of lytic antibody in the mother, however (Ptrend < 0.0001; Table 3).
Effect of HIV Status on KSHV Seropositivity
Table 1 shows KSHV seropositivity according to HIV status. In the 1165 children tested for HIV, KSHV seropositivity was significantly higher in HIV-coinfected children (28.6% vs. 14.6%; P = 0.0005). Similarly, in mothers tested for HIV (n = 1075), KSHV seropositivity was higher with HIV coinfection (41.0% vs. 25.5%; P < 0.0001). In Table 3, 68 (13.7%) of 497 children born to HIV-negative mothers were KSHV-seropositive compared with 32 (20.1%) of 159 children born to HIV-infected mothers (OR = 1.6, 95% CI: 1.0 to 2.6; P = 0.07). Overall, the increased risk for KSHV seropositivity in all children was statistically significant if the mother was also KSHV-seropositive, irrespective of the mother's HIV status. The HIV status of the mother or child did not alter the risk of KSHV infection (χ2 interaction = 0.75, P = 0.38, adjusted for age of mother and HIV status of child).
KSHV Antibody Titers in Mothers and Children According to Their HIV Status
The overall geometric mean (SD range) KSHV antibody levels, as measured by OD, of the mothers were 0.52 (0.10-1.09) for lytic KSHV K8.1 and 0.28 (0.02-0.60) for the latent KSHV ORF73. In HIV-infected mothers, the mean antibody levels for the lytic KSHV K8.1 and latent KSHV ORF73 assays were significantly higher than in the HIV-negative mothers (0.69 [0.20-1.38] vs. 0.47 [0.08-0.99]; P < 0.0001 and 0.35 [0.05-0.71] vs. 0.25 [0.02-0.54]; P < 0.0001, respectively). In children, the mean antibody levels for the lytic KSHV K8.1 and latent KSHV ORF73 were 0.43 (0.16-0.77) and 0.21 (0.06-0.38), respectively. As in their mothers, there was a significant difference in mean antibody levels in children between HIV-infected and HIV-negative subjects for the lytic KSHV K8.1 (0.55 [0.20-1.0] vs. 0.42 [0.1-0.74]; P < 0.0001) and the latent KSHV ORF73 (0.27 [±0.07-0.51] vs. 0.20 [0.06-0.36]; P < 0.0001) assays.
KSHV Seropositivity in Children in Relation to Maternal Kaposi Sarcoma-Associated Herpesvirus Antibody Levels and HIV Status
Table 3 shows the risks for KSHV seropositivity in children aged 1.6 to 10 years for the latent KSHV ORF73 and lytic KSHV K8.1 assays in relation to maternal KSHV antibody levels and HIV status. The risk of KSHV K8.1 seropositivity in children was higher with increasing maternal levels of antibody to KSHV K8.1 (OR = 3.2, 95% CI: 1.6 to 6.1; P trend < 0.0001). The increased risk with increasing maternal K8.1 antibody level was higher if the mother was HIV-negative (Ptrend = 0.0002 vs. Ptrend = 0.09). No increased risk of seropositivity in children was observed with increasing maternal ORF 73 antibody levels (Ptrend = 0.12).
Although the subjects in our study were not randomly sampled from the population, they were recruited from widely distributed geographic locations throughout South Africa, and HIV prevalences in our study were similar to previously reported prevalences.21 Therefore, our results can be interpreted as representative of the prevalence of KSHV in this region. The seroprevalence of KSHV in mothers was nearly 2-fold higher than in children (29.7% vs. 15.9%; see Table 1), which agrees with studies showing an increase in KSHV seropositivity with increasing age and with previous reports on KSHV seroprevalence in South African children and mothers.9,10,22 The risk for KSHV seropositivity was twice as high in children older than 18 months born to KSHV-seropositive mothers compared with children of the same age born to KSHV-seronegative mothers (see Table 2), which, again, is consistent with previous reports.10,15 Notably, and in contrast to a previous study,15 a relatively high proportion of children (12.2%) who were seropositive for KSHV antibodies were born to KSHV-seronegative mothers (see Table 2). Somewhat surprisingly, the risk of KSHV infection in children aged 1.6 to 3 years did not differ by the serostatus of the mother. This is difficult to explain, because one might expect that infants are more likely to have closer contact with their mothers than older children. One possible explanation is that infection with KSHV requires many years of contact with an infected person who may shed virus in saliva only intermittently.
A significant association was observed between the mothers' increasing lytic KSHV K8.1 antibody levels and the risk for KSHV seropositivity in the children. High maternal ORF 73 antibody levels were not associated with increased risk (see Table 3). These findings suggest that mothers with well-controlled infection characterized by high latent but low lytic antibody levels are less likely to be a source of KSHV infection for their children than mothers undergoing frequent reactivation of virus and viral shedding, as characterized by high levels of lytic but not latent antibodies.
KSHV seroprevalence was statistically significantly higher in children and mothers who were HIV-seropositive compared with those who were HIV-seronegative (see Table 1). Also, HIV-positive subjects had significantly higher lytic and latent KSHV antibody levels than HIV-negative subjects. We could not show a clear risk for KSHV infection in children in relation to the mother's HIV status, however. The association between KSHV and HIV coinfection in mothers and transmission of KSHV to children needs to be studied further. Although KSHV titers were higher in HIV-positive mothers compared with HIV-negative mothers, it is not clear whether HIV infection promotes increased shedding of KSHV among mothers, and therefore transmission to children, or whether HIV-infected children are more susceptible to infection by KSHV.
This study confirms that KSHV-infected mothers are a major source of KSHV infection in children and also shows that KSHV infection is considerably higher in HIV-infected subjects in South Africa in contrast to some other African countries, where KSHV infection does not vary by HIV infection status.23-25 Thus, the HIV epidemic may change the pattern of KSHV infection and, subsequently, the incidence of KS in the HIV-positive and HIV-negative populations in South Africa over the next few decades. Longitudinal studies would provide the most useful data to examine the effects of HIV on the transmission of KSHV in this population.
The authors gratefully acknowledge Dr. Anthony B. Lane, Head of the Serogenetics Laboratory, Department of Human Genetics, NHLS, for granting permission to use the stored laboratory samples. They also thank Helen M. Mathabatha and Lettie Bester for their assistance with sample and data collection and Dr. Sam Mbulaiteye for useful discussions.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
coinfection; HIV; human herpesvirus-8; Kaposi sarcoma-associated herpesvirus